Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor in humans. Temozolomide (TMZ) is a critical component of therapy for newly-diagnosed GBM. TMZ is a DNA alkylating agent, methylating the N7 and O6 positions of guanine, and has been used for the treatment of GBM and melanoma. The therapeutic benefit of TMZ depends on its ability to damage DNA and trigger cell death. In addition to cell death, TMZ induced DNA damage can also be repaired, leading to cell survival. The latter outcome results in reduced TMZ efficacy and the development of TMZ resistance. Indeed, almost all GBM patients develop resistance to this drug. Therefore, TMZ resistance is a giant obstacle for the treatment of brain tumors, and it is critically important to determine the molecular mechanisms of acquired TMZ resistance. It is known that O6- methylguanine lesions are repaired by O6-methylguanine-DNA-methytransferase (MGMT); therefore, expression of MGMT confers TMZ resistance. Supporting this idea, multiple clinical studies have indicated that DNA methylation at the MGMT promoter, which results in silencing of MGMT, is associated with prolonged survival of patents receiving both radiation and TMZ treatment. However, even with favorable MGMT promoter hypermethylation, over 40% of patients suffer tumor progression during TMZ therapy, suggesting that mechanisms other than MGMT expression also contribute to TMZ resistance. Our preliminary results indicate that phosphorylation of histone H4 serine 47 (H4S47P) by the Pak2 kinase contributes to the development of TMZ resistance by regulating the expression of MGMT and other genes that confer TMZ resistance. This novel epigenetic mechanism, H4S47P and Pak2-mediated gene regulation, has not been studied in any form of cancer before. Therefore, in this proposal, we will determine how phosphorylation of H4S47 is regulated under TMZ-induced stress, elucidate the molecular mechanisms by which Pak2 and H4S47P contribute to TMZ resistance; and determine to what extent Pak2 and H4S47P levels correlate with the prognosis of primary brain tumors. Together, these studies will reveal a novel epigenetic mechanism by which acquired TMZ resistance is regulated and validate Pak2 as a potential therapeutic target for overcoming TMZ resistance. PUBLIC HEALTH RELEVANCE: Glioblastoma multiforme (GBM), accounting for 52% of all primary brain tumor cases, is the most aggressive type of primary brain tumor. Temozolomide (TMZ) is one of the standard drugs used to treat GBM. However, the efficacy of TMZ is limited by the fact that most patients develop resistance to this drug. Therefore, there is a critical need to address how TMZ resistance is developed. Our preliminary results support the hypothesis that phosphorylation of histone H4 serine 47 (H4S47P) catalyzed by the Pak2 kinase contributes to the development of TMZ resistance in brain tumors. In this proposal, we will employ GBM xenograft models, primary GBM samples and molecular biology techniques to test this hypothesis. These studies will reveal a novel epigenetic mechanism whereby TMZ resistance is developed and potentially identify a novel drug target to combat TMZ resistance, a giant obstacle to successful cancer chemotherapy.